Project Summary C. difficile infection is an extremely debilitating disease with high mortality rates and expensive medical care costs. Current antibiotic therapies such as vancomycin and metronidazole leave patients prone to relapse; therefore, more effective therapeutic approaches are actively sought after. A recently published report from our laboratory demonstrated that a cathelicidin mimic compound CSA13 could inhibit C. difficile primary infection and vancomycin-associated relapse in mice. We found that the oral administration of CSA13 modulated the intestinal microbiome in C. difficile-infected mice. Metabolomic analysis revealed a specific pattern of fecal metabolites associated with the therapeutic effect of CSA13. Oral administration of four metabolites mimicked the protective effect of CSA13. Metabolite treatment, like CSA13 treatment, prevented vancomycin-associated relapse of C. difficile-infected mice. Metabolites may be a potential therapeutic agent against primary C. difficile infection and preventing C. difficile relapse. However, it is necessary to optimize the regimen for maximal efficacy and characterize its impact on C. difficile pathophysiology, intestinal microbiome, and host immune responses. This application proposes to determine (1) whether the metabolites inhibit C. difficile toxin production and spore generation; (2) whether metabolites exert a cytoprotective effect in epithelial cells and suppress toxin-mediated immune responses. We will optimize the dosing regimen of mixed metabolites for maximal protection against CDI. The overall impact of this study will discover a novel way of treating CDI. Specific protective metabolite therapy is safer, more convenient, and more straightforward than fecal microbiota transplantation.